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化学合成H5N1亚型禽流感病毒M2蛋白N端胞外域基因序列3拷贝基因(3M2e),与人结核杆菌hsp70蛋白基因融合,克隆至原核表达载体pET-32a(+),构建表达载体pET-3M2e与pET-3M2e-hsp70。重组质粒转化大肠杆菌BL21,经IPTG诱导重组蛋白获得表达,通过镍亲和层析获得纯化的融合蛋白,利用Western blot鉴定重组蛋白免疫原性。将纯化的重组蛋白免疫20日龄的禽流感非免鸡,以人工合成的M2e与KLH偶联后免疫鸡作为阳性对照,以pET-32a(+)载体蛋白注射组作为阴性对照,初免后2周加强免疫。通过抗M2e抗体检测、细胞病变抑制试验与间接免疫荧光试验评价重组蛋白免疫后产生的体液免疫反应;利用流式细胞分群及细胞因子产量检测评价重组蛋白免疫后产生的细胞免疫反应。加强免疫后4周用100EID50的H9N2亚型AIV攻毒,通过Real-time PCR检测攻毒后3、5、7天免疫鸡泄殖腔棉拭子中病毒的含量。结果表明,重组蛋白3M2e hsp70免疫组能够刺激免疫鸡产生较高的抗体水平及细胞免疫应答,并且攻毒后泄殖腔棉拭子中病毒含量明显降低。
The 3-copy gene (3M2e) of the N-terminal extracellular domain of M2 protein of H5N1 subtype of avian influenza virus was chemically synthesized and fused with the hsp70 protein of Mycobacterium tuberculosis and cloned into the prokaryotic expression vector pET-32a (+) to construct the expression vector pET-3M2e With pET-3M2e-hsp70. Recombinant plasmids were transformed into E. coli BL21 and expressed by IPTG induction of recombinant protein. Purified fusion protein was obtained by nickel affinity chromatography, and the immunogenicity of the recombinant protein was identified by Western blot. The purified recombinant protein was immunized with 20-day-old bird flu non-free chicken. The synthetic chicken M2e conjugate with KLH was used as a positive control and the pET-32a (+) carrier protein injection group as a negative control. 2 weeks to strengthen the immune. The anti-M2e antibody test, cytopathic effect inhibition test and indirect immunofluorescence assay were used to evaluate the humoral immune response induced by the recombinant protein. The cellular immune response induced by recombinant protein immunization was evaluated by flow cytometry and cytokine production assay. Forty weeks after booster immunization, H5N1 subtype AIV was challenged with 100EID50 H9N2 subtype AIV, and the virus content in chickens immunized with chickens was detected by Real-time PCR at days 3, 5 and 7 after challenge. The results showed that the recombinant protein 3M2e hsp70 immune group can stimulate immune chickens to produce higher antibody levels and cellular immune response, and the virus content in the cloaca swab after challenge was significantly reduced.